Method for large scale production of virus antigen

ABSTRACT

The present invention provides improved methods of production of viral antigen on a culture of adherent cells bound to a microcarrier, wherein the methods provide for increased viral antigen yield per culture medium volume. The invention is also directed to a cell culture biomass of adherent cells having increased cell density and microcarrier concentration compared to the respective confluent cell culture.

FIELD OF THE INVENTION

The present invention is directed to improved methods of production ofviral antigen on a culture of adherent cells bound to a microcarrier,wherein the methods provide for increased viral antigen yield perculture medium volume. The invention is also directed to a cell culturebiomass of adherent cells having increased cell density and microcarrierconcentration compared to the respective confluent cell culture.

BACKGROUND OF THE INVENTION

Efficient vaccine production requires the growth of large scalequantities of virus produced in high yields from a host system. Thecultivation conditions under which a virus strain is grown is of greatsignificance with respect to achieving an acceptable high yield of thestrain. Thus, in order to maximize the yield of the desired virus, boththe system and the cultivation conditions must be adapted specificallyto provide an environment that is advantageous for the production of thedesired virus. Therefore, in order to achieve an acceptably high yieldof the various virus strains, a system which provides optimum growthconditions for a large number of different virus is required.

The only process which is economically viable is a reactor processbecause the scale-up can be made appropriate to the market size and thevaccine doses needed. For adherent cells the carrier process with aclassical microcarrier is currently the best choice for large scalecultivation of the cells needed for virus propagation (Van Wezel et al.1967. Nature 216:64-65; Van Wezel et al. 1978. Process Biochem. 3:6-8).Large-scale process production of poliomyelitis virus, Hepatitis AVirus, HSV or Mareck's disease virus on microcarrier has been described(U.S. Pat. No. 4,525,349; Widell et al., 1984. J. Virological Meth.8:63-71; Fiorentine et al., 1985. Develop. Biol. Standard 60:421-430;Griffiths et al., 1982. Develop. Biol. Standard. 50:103-110). Currentprocesses based on microcarrier culture allow production of virus usingfermenter sizes of up to 1200 l.

Caij et al. (1989. Arch. Virol. 105: 113-118) compared production yieldsof virus titre of Hog Cholera Virus on microcarrier cultures andconventional monolayer cultures and found that using the microcarriersystem higher virus yield per volume of medium can be obtained.

Griffiths et al. (1982. Develop. Biol. Standard. 50:103-110) studied theinfluence of the microcarrier concentration on cell growth andproduction of HSV. It was found that an optimal concentration ofmicrocarriers is needed to reach high cell density, which alsoinfluences the virus yield obtained. Higher concentrations ofmicrocarrier in a perfusion system, however, resulted in a cell loss dueto cell layer sloughing off the beads.

The productivity of the virus production process on the microcarriersystem depends on the virus, the cells, the type of microcarrier and thecell density obtained in the system. Higher microcarrier concentrationsin the cell culture allow for higher total cell numbers. However,microcarriers are costly and, in these conditions, cell loss may occurdue to the cell layers sloughing off the beads by the shearing force inthe system. This implies that for higher virus yields a larger volume ofmicrocarrier cell culture is needed, but this increases the efforts thathave to be made for processing and purification such large volumes.

For virus propagation it is important that optimal cell density isreached to obtain maximal virus yield. It is also important to allowefficient adsorption of virus to the cells. In conventional methods,therefore, the volume of the growth medium is reduced prior to infectionto allow adsorption of the virus to the cells in a minimum of culturevolume and for a better virus to cells ratio. However, to obtain optimalvirus propagation, the culture medium volume is again increased afterappropriate adsorption time to allow the cells to maintain viabilityand/or growth. This, however, increases the culture medium volumecomprising cells and/or virus which has the disadvantage that largevolumes have to be processed for further purification of the virus fromthe cells or the cell culture medium.

In the case of an outbreak of a virus infection, it is critical toproduce large amounts of a vaccine in a timely fashion to provideseveral million vaccine doses within a very short period of time.Therefore, a continuing need exists for safe and effective methods toproduce viruses and antigens. Moreover, there is a need for an approachto viral propagation, employing materials that are already available andrequiring a minimal number of time-consuming manipulations, such ashandling of reduced volumes of cell culture medium and facilitatepurification and down stream processing for vaccine production.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide for a method forproduction of virus or viral antigen in a cell culture of adherent cellsbound to microcarrier.

It is also an object of the present invention to provide for a method ofproduction of virus in a small cell culture volume.

It is also an object of the present invention to provide for a cellculture of adherent cells having higher cell density compared to theoriginal cell culture grown to confluence.

It is an object of the invention to provide for a cell culture ofadherent cells bound to microcarrier and having higher cell densitycompared to the original cell culture grown to confluence, wherein thesecells are infected with virus.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with these and other objects, the present inventionprovides methods for production of virus or viral antigen, comprisingthe steps of providing a culture of adherent cells bound to amicrocarrier, growing the cell culture to confluence, infecting thesecells with a virus, wherein the cell density in the cell culture isincreased (i) prior to infection with the virus or (ii) after infectionwith the virus, and incubating the culture of cells infected with thevirus to propagate the virus. The increase of cell density in the cellculture is done by concentration of the cell culture, which includes anincrease of microcarrier concentration in the cell culture.

In general, adherent cells bound to microcarriers need an optimal ratioof microcarrier concentration to cells to reach high cell density. Theincrease of microcarrier concentration in the cell culture theoreticallywould allow to reach a higher cell density per volume of culture medium.However, due to the shearing effects, reduction of feeding sources inthe medium and physiological stress of the cells by increasedmicrocarrier concentration, the carrier concentration in a cell culturesystem is limited to a specific concentration (see also Griffiths et al.1982, supra).

The method of the invention allows the cells to grow under optimalgrowth conditions, including microcarrier concentration, feeding andminimal physiological stress, to reach the maximal cell density for thesystem used.

In the present invention, it is found that reduction of the culturemedium volume prior or after infection with virus, whereby the celldensity and microcarrier concentration in the cell culture biomass isincreased, does not influence the productivity of the cells. Incontrast, it is also surprisingly found that the virus yield obtainedper cell can be increased compared to cells that are maintained at thesame cell density as the original confluent cell culture. This washighly unexpected as due to the increase of the microcarrierconcentration in the cell culture, a reduction of cell viability,sloughing of the cells from the microcarriers and physiological stressdue higher cell density and during virus production would have beenexpected.

The method of the invention allows to reduce the culture medium volumethat has to be processed during the further purification process of thevirus, while simultaneously the productivity of virus per cell issimilar or even increased compared to the original cell culture. Thesystem can be scaled-up to 6000 l fermenter volume, which makes theprocess for virus production for vaccines more efficient and lesstime-consuming.

According to one embodiment of the method the anchorage-dependent cellsare selected from the group of adherent cells of VERO, BHK, CHO, RK,RK44, RK13, MRC-5, MDCK, CEF or diploid monolayer cells as described byReuveny et al. (1985. Develop. Biol. Standard. 60:243-253) and otherswell known in the art.

The adherent cells bound to a microcarrier can be grown in conventionalculture medium containing serum. According to a preferred embodiment ofthe invention the cells are grown in serum free or serum and proteinfree medium as described by Kistner et al. (1998. Vaccine 16: 960-968),Merten et al. (1994. Cytotech. 14:47-59), Cinatl. et al. (1993. CellBiology Internat. 17:885-895), Kessler et al. (1999. Dev. Biol. Stand.98:13-21), WO 96/15231, U.S. Pat. No. 6,100,061 or any other serum freeor serum and protein free medium known in the art. The cells arepreferably grown from the ampoule to the large scale to the biomass inserum free or serum and protein free medium.

According to one embodiment of the invention the culture of adherentcells bound to a microcarrier are grown to confluence and infected witha virus after increase of cell density and microccarrier concentrationof cell biomass of the confluent cell culture.

According to one embodiment of the invention the culture of adherentcells bound to a microcarrier is grown to confluence and infected with avirus prior increase of cell density and microccarrier concentration ofthe confluent biomass. In any case, if either the cell culture havinghigher cell density and microcarrier concentration per volume isinfected prior or after concentration of the culture, the cell density,microcarrier concentration in the biomass is kept constant during viruspropagation and production process, while the volume of the medium isnot increased again.

The method used to increase the cell density and microcarrierconcentration in the cell culture biomass, either uninfected or infectedwith a virus, can be any method known in the art to concentrate a cellculture. This can be done by methods like, e.g. sedimentation,centrifugation, filtration, concentration with a perfusion device, likea sieve, that allows the reduction of working volume, or by pooling 2 ormore bioreactor systems.

The cell culture density and microcarrier concentration of the cellculture grown to confluence are increased, wherein the increase shouldbe at least 1,3-fold compared to the original biomass grown toconfluence. The cell density of the original starting cell culture thathas been grown to confluence can be between about 0.6×10⁶ and about7.0×10⁶ cells/ml. In this case, the biomass having increased celldensity compared to the starting culture biomass can have a cell densitybetween at least 0.8×10⁶ and at least 9.0×10⁶ cells/ml.

The microcarrier concentration in the start cell culture is preferablyin the range of about 0.5 g/l to about 7.0 g/l. The concentration of themicrocarrier after concentration of the confluent biomass is preferablyin the range of about 0.65 g/l and about 21 g/.

The microcarrier used according to the method of the invention ispreferably selected from the group of microcarriers based on dextran,collagen, polystyrene, polyacrylamide, gelatine, glass, cellulose,polyethylene and plastic and those described by Miller et al. (1989.Advances in Biochem Eng./Biotech. 39:73-95) and described in Butler(1988. In: Spier & Griffiths, Animal cell Biotechnology 3:283-303).

According to one embodiment of the method of the invention the virus isselected from the group of Influenza virus, Ross River Virus, HepatitisA Virus, Vaccinia Virus and recombinant Vaccinia Virus, Herpes SimplexVirus, Japanese encephalitis Virus, West Nile Virus, Yellow Fever Virusand chimeras thereof, as well as Rhinovirus and Reovirus. It is withinthe knowledge of one skilled in the art to select an adherent host celland the virus susceptible to this host and to use the method of theinvention to obtain increased virus yield of the desired virus.

It is within the knowledge of one skilled in the art to select therespective microcarrier type, the microcarrier concentration in thestarting culture, the adherent cells susceptible to the virus, and themedium and optimal growth conditions, like oxygen concentration,supplements of the medium, temperature, pH, pressure, steering speed andfeeding control, to obtain a confluent cell culture biomass which can beused to obtain a cell biomass having increased cell density andmicrocarrier concentration according to this method. The cell culturehaving higher cell density biomass can be used then for effective viruspropagation and production. After the cell culture has reachedconfluency, the method of the invention allows to obtain a cell culturehaving an increased cell density of microcarrier concentration of atleast 1,3-fold up to 10 fold and obtain higher virus yield per culturevolume due i) reduced culture volume and ii) increased productivity percell.

The virus production process and the time span for production depend onthe system used. The maximal virus yield reachable in the respectivesystem can be determined by standard methods. When maximal virusproduction yield is reached, the virus and/or cells comprising the virusare harvested. The method of the invention, therefore, further comprisesa step of harvesting the virus propagated and produced.

Another aspect of the invention provides for a method for production ofpurified virus or virus antigen comprising the steps of providing aculture of adherent cells bound to a microcarrier, growing the cellculture to confluence, infecting the culture of cells with a virus,wherein the cell density in the cell culture is increased (i) prior toinfection with the virus or (ii) after infection with the virus,incubating said culture of cells infected with said virus to propagatesaid virus (f) harvesting the virus produced and (g) purifying saidvirus harvested.

Dependent on the nature of the virus used for infection and propagation,the virus produced is either found in the supernatant of the cellculture and/or associated with the cellular biomass. Lytic viruses, suchas Influenza virus, lyse the cells after appropriate time afterinfection and the virus is released into the cell culture medium. Thevirus produced and released in the cell culture medium can be separatedfrom the cellular biomass or other cell fragments by conventionalmethods, such as centrifugation, including ultracentrifugation, densitygradient centrifugation, microfiltration, ultrafiltration, ion exchangechromatography etc. and purified.

Non-lytic viruses propagate within the cells and are still associatedwith the cells of the biomass. These viruses can be harvested bycollecting the biomass, lysing the cells by conventional methods, suchas treating the cells with a detergent, heat, freeze/thawing,sonication, French-press or other cell lysing methods. The virusesreleased from the cells are harvested, concentrated and purified. Thepurification of the virus can be done by any method known in the art,such as ultrafiltration, ion exchange chromatography or isopygniccentrifugation etc.

Influenza virus can be propagated on cell lines, including the mostefficient MDCK cells, as well as on the cell line that has been licensedfor use in the manufacture of human vaccines, Vero cells. Large scaleproduction of Influenza virus in serum free or serum free and proteinfree medium on a mammalian cell culture on microcarrier beads in abioreactor and the development of a Influenza virus vaccine has beendescribed (Merten et al., 1999, Dev. Biol. Stand. 98: 23-37; Kistner etal., 1998. Vaccine 16:960-968; Kistner et al. 1999, Dev. Biol. Stand.98:101-110 and WO 96/15231.

According to one aspect, the invention provides for a method forproduction of Influenza virus, comprising the steps of providing aculture of adherent cells bound to a microcarrier, growing the cellculture to confluence, infecting the cells with an Influenza virus,wherein the cell density in the cell culture is increased (i) prior toinfection with the virus or (ii) after infection with the virus,incubating the culture of cells infected with said Influenza virus topropagate the virus. The cells infected with Influenza virus can be VEROor MDCK cells, or any cell that is susceptible to Influenza virus.According to a preferred embodiment of the invention, VERO cells areused and infected with Influenza virus. According to a preferredembodiment, the VERO cells are grown in serum free or serum and proteinfree medium from the original ampoule to the biomass. The VERO cellsbound to the microcarrier are grown in the respective medium toconfluence and cell density and microcarrier concentration is increasedat least 1,3 fold. The cells can be infected with Influenza virus eitherprior or after increase of cell density of culture volume. Afterincubation of the infected high cell density biomass and production ofvirus, the Influenza virus or Influenza virus antigen produced isharvested. The harvested virus is further purified by a method known inthe art, such as described in Kistner et al. 1998 (supra) or U.S. Pat.No. 6,048,537.

Another aspect of the invention provides for a cell culture biomass ofadherent cell bound to microcarrier having high cell density, whereinthe cell density biomass of the cells in the cell culture is at least1,3-fold compared to a cell culture that has been grown to confluence.The culture of adherent cells bound to a microcarrier are cells selectedfrom the group of anchorage-dependent cells of VERO, BHK, CHO, RK, RK44,RK13, MRC-5, MDCK, CEF or diploid monolayer cells. The cell culturebiomass having high cell density is preferably a culture of VERO cells.

According to a preferred embodiment of the invention the cell culturebiomass is grown in serum free medium and does not comprise anysubstances or agents derived from serum. According to another preferredembodiment the biomass is serum and protein free and does not compriseany serum derived substances or proteins added to the medium.Preferably, the cells have been grown in serum free or serum and proteinfree medium from the original ampoule to the biomass. The biomass havinghigh cell density is maintained in serum free or serum and protein freemedium during virus propagation and production process.

According to another embodiment of the invention the cells of thebiomass having higher cell density compared to the cell culture that hasbeen grown to confluence are infected with a virus. The cell density andvolume of the culture medium of the high cell density biomass infectedwith virus is maintained during the virus propagation process.

Another aspect of the invention provides for a cell culture biomass ofVERO cells bound to a microcarrier, wherein the biomass and the celldensity of the VERO cells in said cell culture is at least 1,3-foldcompared to a VERO cell culture that has been grown to confluence. Thecell culture has also a higher microcarrier concentration compared tothe cells gown to confluence.

According to a preferred embodiment of the invention the cell culturebiomass having higher cell density is a biomass of VERO cells.Preferably, the cells are grown in serum free medium and the biomass isserum free. According to another preferred embodiment of the inventionthe biomass culture is serum and protein free.

Another aspect of the invention provides for a cell culture biomass of acell culture of adherent cells bound to a microcarrier infected with avirus, wherein the biomass of the infected cells in said cell culture isat least 1,3-fold compared to a cell culture that has been grown toconfluence prior to infection, and has higher cell density. According toone embodiment the cell culture biomass of cells is serum free.According to another preferred embodiment of the invention the cellculture biomass is serum and protein free. The cells are preferably VEROcells. This cell density of the high cell density biomass infected withvirus is not decreased during the virus propagation process.

Another aspect of the invention provides for a cell culture biomass ofVERO cells bound to microcarrier and having a high cell density bound toa microcarrier, wherein the biomass of the VERO cells in said cellculture is at least 1,3-fold compared to a VERO cell culture that hasbeen grown to confluence, wherein the VERO cells are infected withvirus. The VERO cells are infected with a virus selected from the groupof Influenza virus, Ross River Virus, Hepatitis A Virus, Vaccinia Virusand recombinant derivatives thereof, Herpes Simplex Virus, Japaneseencephalitis Virus, West Nile Virus, Yellow Fever Virus and chimericthereof, Rhinovirus and Reovirus.

According to one another aspect, the invention provides a cell culturebiomass of VERO cells bound to microcarrier and having high cell densitysaid cells being infected with a Influenza virus, wherein the biomass ofthe VERO cells in said cell culture is at least 1,3-fold compared to aVERO cell culture that has been grown to confluence.

According to one another aspect, the invention provides a cell culturebiomass of VERO cells bound to microcarrier and having high cell densitysaid cells being infected with a Ross River virus, wherein the biomassof the VERO cells in said cell culture is at least 1,3-fold compared toa VERO cell culture that has been grown to confluence.

According to one another aspect, the invention provides a cell culturebiomass of VERO cells bound to microcarrier and having high cell densitysaid cells being infected with a Hepatitis A virus, wherein the biomassof the VERO cells in said cell culture is at least 1,3-fold compared toa VERO cell culture that has been grown to confluence.

Having now generally described this invention, the same will beunderstood by reference to the following examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLE 1 Virus Antigen Production on Concentrated Vero Cell Biomass

a) Growth of Cell Culture

VERO cells (African Green Monkey, Cercopthecus aethiops, kidney) wereused as a production cell line. The cells have been obtained from theAmerican Type Cell Culture Collection, Rockville, Md. at a passagenumber 124 under the designation ATCC CCL 81. The cells were adapted togrow in serum free or serum and protein free medium as described inKistner et al., 1998 (supra), WO 96/15231 or U.S. Pat. No. 6,100,061.For growth in serum free medium a basal DMEM HAM's F12 mediumsupplemented with inorganic salts, amino acids, sodium bicarbonate (2g/l) and yeast or soy bean extract (0.1 to 10 g/l) is used. The workingcell bank was prepared without the use of any animal derived mediumcomponents.

Cells of the working cell bank were expanded in T-flasks and rollerbottles with a split ratio of 1:6 or 1:8. Further propagation of thecells was performed in a 100 l stirred tank bioreactor using Cytodex®microcarrier as attachment substrate. The cells were grown at 37° C. for6-8 days. The culture conditions of oxygen saturation 20%+/−10% andpH7.25+/−0.35 were kept constant. At the end of biomass production whencell have reached confluence growth, one part of the biomass reactorvolume was concentrated two-fold by sedimentation and the cell densityof the unconcentrated and concentrated cell culture was determined.

b) Determination of Cell Density of Biomass

The cell number of the biomass of the cell culture at the end of biomassproduction was determined either by trypsinization of the cells andcounting with a CASY® cell counter (method A) as described by Schärfe etal. (1988. Biotechnologie in LaborPraxis 10:1096-1103) or by citric acidand crystal violet treatment followed by counting with a haemocytometer(method B) as described by Sanford et al. (1951. J. Natl. Cancer Inst.11:773-795). The cell density and carrier concentration for Vero cellsat the end of biomass production and after concentration of theconfluent biomass (prior infection) were calculated by method A and B.The data are shown in Table 1.

TABLE 1 Determination of cell number in a confluent cell culture at theend of biomass production and after concentration of confluent cellculture Biomass production Concentrated Biomass Carrier Concentrationg/l 5.0 10.0 Cell Density cells/ml 4.6 × 10⁶  9.2 × 10⁶ (method A) CellDensity cells/ml 5.6 × 10⁶ 11.2 × 10⁶ (method B)

EXAMPLE 2 Comparison of Virus Antigen Production of a Confluent Biomassand a Concentrated Confluent Biomass

Vero cells with a defined passage number were thawed from liquidnitrogen and passaged in roux and roller bottles to produce sufficientcells to inoculate a 1.5 liter bioreactor. After reaching confluencywith a final cell density of 1.5×10⁶ cells/ml the cells were trypsinizedand transferred to a 10 liter bioreactor. This in turn is used as aninoculum for a 100 liter bioreactor having a microcarrier concentrationof 1.5 g/l. Starting from a working cell bank ampoule containing 10⁷cells about 30 generations are needed to reach the final confluent Verocell biomass. The culture was grown to reach confluency with a finalcell density of 1.9×10⁶/ml. Prior to virus infection, two 10 literbioreactor systems were loaded with cell culture biomass, havingdifferent total cell numbers. Fermenter A is loaded with 1.9×10¹⁰ cells,and fermenter B with a total cell number of 3.8×10¹⁰. To achieve higherbiomass and carrier concentration to load fermenter B the cell culturegrown to confluency was concentrated by sedimentation of the biomass toreach a two-fold concentration. Fermenter A contains 100% and fermenterB 200% cell biomass of the original cell culture grown to confluency.

a) Production of Influenza Virus

The cell culture in fermenter A and B were infected with Influenza virusstrain H3N2 A/Sydney/5/97 with a m.o.i of 0.01. Identical processparameters of 32° C., pO₂ of 20% and pH 7.1 were applied. To activateInfluenza virus for virus propagation a protease, such as trypsin,pronase or a trypsin-like part thereof, was added.

The virus antigen productivity of the two different cell cultures offermenter A and B containing different biomass concentrations wasdetermined and compared on the basis of Influenza virus titer (HAU/ml)and the antigen content (density gradient purified antigen). The peakarea corresponds to the total antigen concentration at the end of thelytic cycle at day 3 after infection. The data is shown in Table 2.

TABLE 2 Determination of Influenza Virus titer and antigen in aconfluent VERO cell culture and concentrated confluent VERO cell biomassFermenter A B Carrier Concentration 1.5 g/l 3.0 g/l Cell Densitycells/ml 1.90 × 10⁶ 3.80 × 10⁶ (method B) HAU/ml 640 2560 Peak Area(rel. Units) 83.3 (100%) 412.3 (495%)b) Production of Ross River Virus

VERO cells were propagated as described above to confluency with a finaldensity of 1.6×10⁶ cells/ml. Prior to virus infection two 50 lbioreactor systems were loaded with cell culture biomass, havingdifferent total cell numbers. Fermenter A is loaded with 1.6×10⁶cell/ml, and fermenter B with 2.3×10⁶ cells/ml, which is a 1,5 foldconcentration of c confluent cell culture biomass. Fermenters A and Bwere infected with Ross River Virus and virus antigen productivity offermenter A and B were determined as described above. Table 3 shows theresults of virus yield obtained by using different concentrations ofbiomass for virus propagation.

TABLE 3 Determination of Ross River Virus titer and antigen productionFermenter A B Carrier Concentration g/l 1.5 2.25 Cell Density (×10⁶cells/ml) 1.6 2.3 Virus titer (log TCID₅₀) 8.71 8.95 Virus titer pfu/10⁶cells (×10⁶) 321 388 Yield (%) 100 121

EXAMPLE 3 Virus Antigen Production on Concentrated Biomass of RK-Cells

a) Growth of Cell Culture

Rabbit kidney cells RK-13 or a complementing derivative thereofRK-D4R-44 as described by Holzer et al. (1997. J. Virol. 71:4997-5002)were used as production cell lines. Cells were grown in conventionalmedium containing 2% serum.

Cells from the working cell bank were expanded in T-flasks and rollerbottles with a split ratio of 1:6. Further propagation of the cells wasdone in a 10 l stirred tank bioreactors using Cytodex200 (Pharmacia)microcarriers as attachment substrate.

b) Production of Defective Vaccinia Virus

After the RK-13 or RK-D4R-44 cells have reached confluence and finalcell density in the tank bioreactors, the biomass was infected withVaccinia Virus WR or defective Vaccinia Virus vD4-ZG#2 as described byHolzer et al. 1997 (supra) with a m.o.i. of 0.01. After infection, two10 l bioreactor systems were loaded with the infected cell culturebiomass, having different total cell numbers. Fermenter A is loaded with1.2×10¹⁰, and fermenter B with a 2.4×10¹⁰. To achieve higher biomass andcarrier concentrations for fermenter B, the infected cell culture grownto confluence was concentrated by sedimentation of the biomass to reachhigher concentration. Fermenter A contains 100% and fermenter B 200%cell biomass of the original cell culture grown to confluence. The virusantigen productivity of the two different cell culture fermenters A andB containing different biomass concentrations per volume of medium ofinfected cells was determined. The results are summarized in Table 4.

TABLE 4 Determination of Vaccinia Virus titer on RK-cells Fermenter A BCarrier Concentration g/l 1.5 2.5 Cell Density (×10⁶ cells/ml) 1.2 2.4Virus titer pfu/10⁶ cells (×10⁶) 0.8 1.3 Yield (%) 100 162

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference for all purposes.

1. A method for production of virus comprising viral antigen, comprisingthe steps of (a) providing a culture of adherent cells bound to amicrocarrier; (b) growing the cell culture to confluence; (c) infectingthe cells with a virus; (d) incubating said culture of cells infectedwith said virus to propagate said virus, wherein the cell density of thebiomass of the cell culture grown to confluence is increased byreduction of working volume (i) prior to step (c) or (ii) after step (c)while the cells are bound to said microcarrier, and wherein the celldensity is maintained at the increased cell density during step (d) ascompared to the biomass grown to confluence during step (b); and (e)harvesting the virus produced.
 2. The method according to claim 1,wherein the density of the cell culture grown to confluence is increasedat least about 1.3 fold.
 3. The method according to claim 1, wherein thecell density of the cell culture grown to confluence is between about0.6×10⁶ and about 7.0×10⁶ cells/ml.
 4. The method according to claim 1,wherein the microcarrier is selected from the group consisting ofmicrocarriers made of dextran, collagen, polystyrene, polyacrylamide,gelatine, glass, cellulose, polyethylene and plastic.
 5. The methodaccording to claim 1, wherein the microcarrier concentration in theculture of cells of step (a) is between about 0.5 g/l and about 14 g/l.6. The method according to claim 1, wherein said cells are selected fromthe group consisting of adherent cells of VERO, BHK, CHO, RK, RK44,RK13, MRC-5, MDCK, CEF and diploid monolayer cells.
 7. The methodaccording to claim 1, wherein said cells bound to a microcarrier aregrown in serum free medium.
 8. The method according to claim 1, whereinsaid cells bound to a microcarrier are grown in serum and protein freemedium.
 9. The method according to claim 1, wherein the virus isselected from the group consisting of Influenza virus, Ross River Virus,Hepatitis A Virus, Vaccinia Virus and recombinant Vaccinia Virus, HerpesSimplex Virus, Japanese encephalitis Virus, West Nile Virus, YellowFever Virus and chimeras thereof, Rhinovirus and Reovirus.
 10. A methodfor production of purified virus or virus antigen comprising the stepsof: (a) providing a culture of adherent cells bound to a microcarrier;(b) growing the cell culture to confluence; (c) infecting the culture ofcells with a virus; (d) incubating said culture of cells infected withsaid virus to propagate said virus; (e) harvesting the virus produced;and (f) purifying said virus harvested, wherein the cell density of thebiomass of the cell culture grown to confluence is increased byreduction of working volume (i) prior to step (c) or (ii) after step (c)while the cells are bound to said microcarrier, and wherein the cellculture is maintained at the increased cell density during step (d) ascompared to the biomass grown to confluence during step (b).
 11. Themethod according to claim 10, wherein the virus produced is harvestedfrom the cell culture supernatant.
 12. The method according to claim 10,wherein the virus produced is harvested from the cell biomass.
 13. Amethod for production of Influenza virus comprising viral antigen,comprising the steps of: (a) providing a culture of adherent cells boundto a microcarrier; (b) growing the cell culture to confluence; (c)infecting the cells with an Influenza virus; (d) incubating said cultureof cells infected with said Influenza virus to propagate said virus,wherein the cell density of the biomass of the cell culture grown toconfluence is increased by reduction of working volume (i) prior to step(c) or (ii) after step (c) while the cells are bound to saidmicrocarrier, and wherein the cell culture is maintained at theincreased cell density during step (d) as compared to the biomass grownto confluence during step (b); and (e) harvesting said Influenza virusproduced.
 14. The method according to claim 13, wherein said cells areVERO cells.
 15. The method according to claim 13, wherein said cells areMDCK cells.
 16. The method according to claim 13, wherein said cellsbound to a microcarrier are grown in serum free medium.
 17. The methodaccording to claim 13, wherein said cells bound to a microcarrier aregrown in serum and protein free medium.
 18. The method according toclaim 13, wherein the cell culture grown to confluence is increased atleast about 1.3 fold.
 19. The method according to claim 1, wherein thecell culture in step (d) is maintained for at least three days.
 20. Themethod according to claim 9, wherein the virus is Influenza virus. 21.The method according to claim 9, wherein the virus is Ross River Virus.22. The method according to claim 9, wherein the virus is selected fromthe group consisting of Vaccinia Virus and recombinant Vaccinia Virus.23. The method according to claim 9, wherein the virus is Japaneseencephalitis Virus, West Nile Virus, Yellow Fever Virus or chimerasthereof.
 24. The method of claim 1, further comprising the step ofpurifying the virus or viral antigen.
 25. The method of claim 13,further comprising the step of purifying the Influenza virus or viralantigen.